Download Ryanodine Myopathies Without Central Cores-Clinical

Pediatric Neurology 51 (2014) 275e278
Contents lists available at ScienceDirect
Pediatric Neurology
journal homepage: www.elsevier.com/locate/pnu
Clinical Observations
Ryanodine Myopathies Without Central CoresdClinical,
Histopathologic, and Genetic Description of Three Cases
João Rocha MD a, *, Ricardo Taipa MD b, Manuel Melo Pires PhD b,
Jorge Oliveira MSc c, Rosário Santos MSc c, Manuela Santos MD d
a
Department of Neurology, Hospital de Braga, Braga, Portugal
Neuropathology Unit, Centro Hospitalar do Porto, Porto, Portugal
c
Molecular Genetics Unit, Centro de Genética Médica Dr. Jacinto Magalhães, Centro Hospitalar do Porto, Porto, Portugal
d
Neuromuscular Diseases Unit, Department of Pediatric Neurology, Centro Hospitalar do Porto, Porto, Portugal
b
abstract
BACKGROUND: Mutations in ryanodine receptor 1 gene (RYR1) are frequent causes of myopathies. They classically
present with central core disease; however, clinical variability and histopathologic overlap are being increasingly
recognized. PATIENTS: Patient 1 is a 15-year-old girl with mild proximal, four-limb weakness from age 5, presenting
with a progressive scoliosis starting at age 10. Patient 2 is an 18-year-old girl with progressively worsening muscle
hypotrophy and mild proximal, four-limb weakness. She developed a rapidly progressive scoliosis from age 11 and
needed surgical treatment at age 14 years. Patient 3 is an 11-year-old boy with moderate proximal limb weakness
and progressive neck flexor weakness, first noticed at age 2. Muscle biopsies revealed type 1 fiber predominance
(Patients 1 and 2) or abnormal type 1 fiber uniformity (Patient 3). Different RYR1 variants were identified in all
patients. In Patients 1 and 3, these changes were validated as being pathogenic. CONCLUSIONS: These patients
illustrate early-onset, progressive myopathies with predominant axial involvement. Histopathologic findings
were abnormal but not specific for a diagnosis, particularly central core myopathy. Genetic testing helped
broaden the range of phenotypes included in the RYR1-related myopathies. Our patients reinforce the need to
recognize the broad histopathologic variability of RYR1-related myopathies and sometimes lack of pathognomonic findings that may reduce the diagnostic threshold of this disease. We suggest that the predominance of
type 1 fibers and involvement of axial muscles may be an important element to consider the RYR1 gene as
candidate.
Keywords: ryanodine receptor, RYR1, myopathy, muscle biopsy
Pediatr Neurol 2014; 51: 275-278
Ó 2014 Elsevier Inc. All rights reserved.
Introduction
The ryanodine receptor 1 gene (RYR1) encodes the main
Ca2þ channel in the skeletal muscle’s sarcoplasmic reticulum, having a crucial role in muscle excitation-contraction
mechanisms.1 More than 300 distinct mutations in RYR1
(19q13.1) have been reported.2 Classically, they have been
Article History:
Received February 10, 2014; Accepted in final form April 24, 2014
* Communications should be addressed to: Dr. Rocha; Department of
Neurology; Hospital de Braga; Sete Fontes, S. Victor; 4710-243 Braga,
Portugal.
E-mail address: [email protected]
0887-8994/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.pediatrneurol.2014.04.024
associated with autosomal dominant central core disease
(MIM #117000) and malignant hyperthermia susceptibility.
Clinical features have been described as globally nonprogressive and include congenital hypotonia and minor
weakness with proximal lower limb predominance (pelvic
girdle). Sometimes, axial involvement without respiratory
compromise, motor milestones delay, or skeletal malformations (hip dislocation, scoliosis, and foot deformities)
may be observed.3 Throughout the years, other phenotypes
have been attributed to RYR1 mutations, including King
Denborough syndrome, limb-girdle myopathy, core-rod
myopathy, bent spine myopathy, exertional or drugassociated myalgia, and rhabdomyolysis and asymptomatic creatine kinase elevation. A marked interpersonal and
276
J. Rocha et al. / Pediatric Neurology 51 (2014) 275e278
even intrafamilial variation in phenotype has been evident
in previous reports.3-6 Typical muscle histopathology includes type 1 muscle fibers with central amorphous areas of
sarcomeric disorganization characterized by the absence of
mitochondria and of oxidative enzymatic activity (“cores”).7
In recent years, there has been an increased awareness of a
marked clinical variability and histopathologic overlap with
other myopathies, precluding a direct diagnosis.3,8 These
phenoptypes include myopathies with multiminicores and
atypical cores, centrally placed nuclei, cores and nemaline
rods, congenital fiber-type disproportion, and marked predominance or uniformity of type 1 fibers.7,8
Although RYR1 gene analysis was initially restricted to
hotspot regions because of its large size (106 exons),
sequencing all the exonic regions of the gene is now recommended, especially when patients have atypical muscle
phenotypes, which are difficult to correlate with the genotype, or in sporadic cases. The broader analysis of RYR1 gene
has brought the molecular diagnosis of these patients to a
new level of complexity because several new sequence
variants are often found, frequently described as unclassified variants, making the genetic diagnosis problematic.
We report three independent pediatric patients with
early-onset myopathies with nonspecific histopathology
and highlight some clinical characteristics that can lead to
the diagnosis; in two instances, mutations were identified
in RYR1 gene.
presence of a heterozygous mutation c.14677C>T (p.Arg4893Trp) in
exon 102 (hotspot 3) (Fig 3). This mutation was first identified by
Monnier et al. in two distinct families with autosomal dominant (AD)
transmission. Similarly, the mutation was also found in our patient’s
mother and uncle suggesting an AD inheritance pattern.
Patient 2
An 18-year-old girl with an unremarkable family history had been
monitored by the Neuropediatrics Department from 12 years of age. She
was described as hypotonic since birth and had mildly delayed motor
milestones. Progressive fatigue and weakness after physical activity was
evident since early childhood, and at age 11, she began developing a
rapidly progressive, severe scoliosis requiring surgical correction and
noninvasive ventilation by age 14. On examination, she had global
muscle hypotrophy and low weight and stature. She also has an arched
palate and axial weakness with involvement of neck flexors, narrow
thorax, and dorso-lombar scoliosis (Fig 1B). Low-grade proximal and
slowly progressive upper and lower limb weakness with joint retractions
has been observed during follow-up. Muscle biopsy at 12 years of age
revealed increased variability in fiber diameter with frequent fiber atrophy and occasional fiber hypertrophy. There was some endomysial
fibrosis and adipose substitution but with preserved internal structure
(Fig. 2, P2). Marked type 1 fiber predominance was also noted. Molecular
study of the RYR1 gene revealed a heterozygous missense variant with
unknown significance c.7843C>T (p.Arg2615Cys) in exon 49 (hotspot 2)
(Fig 3). This previously unreported variant affects a highly conserved
residue. RYR1 exonic regions were fully sequenced, and her asymptomatic father was found to carry the same variant. The muscle complementary DNA study, restricted to the RYR1 transcript region where the
variant is located, demonstrated biallelic expression because the
c.7843C>T was detected in heterozygosity.
Patient Descriptions
Patient 3
Patient 1
A 15-year-old girl with a family history of “lower limb weakness”
(affecting her mother, maternal uncle, and grandfather) was first
observed in the Neuropediatrics Department at age 11 with a slight delay
in motor milestones but stable development up to 10 years of age. At this
point, she started developing a scoliosis that has progressed to a severe
form. Surgical treatment has been proposed but not yet performed.
Physical examination revealed a right ptosis, bilateral facial palsy, and
arched palate. She had considerable axial weakness with neck flexor
involvement, dorso-lumbar scoliosis (Fig 1A), and a moderate, proximal,
nonprogressive upper and lower limb weakness. A muscle biopsy performed when she was 6 years old revealed normal muscle fiber size
variability and preserved internal structure but marked type 1 fiber
predominance (Fig 2, P1). Molecular study of the RYR1 gene revealed the
An 11-year-old boy with an unremarkable family history, first
observed in the Neuropediatrics Department at age 10, exhibited a minor
delay in motor milestones from 2 years of age. On physical examination,
he presented with a bilateral facial palsy, arched palate, and axial
weakness with neck flexor weakness and a narrow thorax (Fig 1C). He
had a moderate-grade proximal and nonprogressive upper and lower
limb weakness. His muscle biopsy, performed at 10 years of age had
normal fiber size variability with global preservation of internal structure and only slight central dimming on oxidative stain but no cores. He
had abnormal type 1 fiber uniformity (Fig 2, P3). Molecular RYR1
revealed three sequence variants: c.4711A>G (p.Ile1571Val) in exon 33;
c.10097G>A (p.Arg3366His in exon 67; c.11798G>A; p.Tyr3933Cys) in
exon 86. These variants have been previously described in the same
allele.3 A heterozygous mutation c.14537C>T (p.Ala4846Val) in exon 101
FIGURE 1.
Pattern with marked axial involvement: (A) Patient 1 vertebral column x-ray revealing dorso-lombar scoliosis; (B) Patient 2 vertebral column x-ray
revealing severe dorso-lombar scoliosis; (C) Patient 3 lateral profile with narrow thorax.
J. Rocha et al. / Pediatric Neurology 51 (2014) 275e278
277
FIGURE 2.
Muscle biopsy histopathologic features. (A) Hematoxylin and eosin stain; (B) Nicotinamide adenine dinucleotide stain; and (C) Adenosine triphosphatase 4,35
stain. P1: (A) normal muscle fiber size variability; (B and C) internal structure is normal, but marked type 1 fiber predominance is observed; P2: (A) some atrophy
and occasional fiber hypertrophy and endomysial fibrosis with scanty adipose replacement; (B and C) normal internal structure with type 1 fiber predominance;
P3: (A) normal fiber size variability; (B and C) internal structure is normal and slight central dimming on oxidative stain, but no cores are observed, and type 1
fiber uniformity. Scale bar: 100 mm.
(hotspot 3) was also present, previously reported as pathologic9 (Fig 3).
The parents are awaiting screening to assess the possibility of autosomal
recessive inheritance.
All analytical studies were normal in the three cases, including
muscle creatine kinase.
Discussion
Our patients illustrate early-onset myopathies with
prominent progressive axial involvement, variable clinical
severity, and progressive course. The first two patients
rapidly evolved to severe scoliosis during the second decade
of life. Patient 3 has not developed scoliosis, having only
marked neck flexor weakness, but he is the youngest of the
three individuals, having only started the second decade of
life. Histopathologic examination revealed changes suggestive of but not classical for RYR1-related myopathy.
All patients had marked type 1 fiber predominance
without typical “cores,” a feature recently recognized to be
FIGURE 3.
Molecular study of RYR1 gene: image reveals sequences’ electropherograms containing mutations (all variants are heterozygous).
278
J. Rocha et al. / Pediatric Neurology 51 (2014) 275e278
associated with RYR1 myopathies.3,10 Noncore RYR1-related
myopathies may be more frequent than initially thought
and are probably underdiagnosed. Amburgey et al.4 stated
that 49% of recessive cases in their study (n ¼ 106) were
noncore myopathies. In these patients, histopathologic
presentation evolves during the course of the disease and
the typical central cores may only appear in later stages.4,7,11
Patient 3 may be an example of such an evolving course
where there seems to be a central dimming on oxidative
stains, without a clear core formation. Endomysial fibrosis
and adipose substitution, as found in the biopsy of Patient 2,
are characteristics more commonly associated with dystrophies and are less specific traits, but they have also been
described in RYR1-related myopathies.7,12
Concerning molecular analysis, Patients 1 and 3 have
RYR1 variants previously recognized as pathogenic. However, Patient 2, the most severely affected of our three cases,
has a heterozygous missense variant of unknown significance (p.Arg2615Cys) in hotspot 2 of the ryanodine receptor
gene. This variant, previously unreported in the literature
and in sequence variant databases, affects a highly
conserved residue and is of paternal origin. RYR1 exonic
regions were fully sequenced, and no other mutation was
found. Although the patient’s father is an asymptomatic
carrier of the same mutation, we cannot completely exclude
its pathologic role, because high inter- and intra-familiar
phenotype variability, ranging from asymptomatic to
severely affected individuals, has been described with other
mutations in this disease.13 Epigenetic allele silencing of the
RYR1 gene due to imprinting could also provide an explanation for our case.14 However, this hypothesis was
excluded because we have detected biallelic expression of
RYR1 gene in complementary DNA obtained from muscle,
demonstrating heterozygous expression. In the first case,
transmission is compatible with an autosomal dominant
inheritance, in contrast with Patient 3, where autosomal
recessive inheritance is likely. This assumption was made
because this sporadic case has four missense variants, three
of which have been previously described in the same allele,
and where p.Ile1571Val and p.Tyr3933Cys were considered
as mutations.3
Our report highlights the phenotypic variability of RYR1
myopathies, which may lack pathognomonic histopathologic findings, or these may overlap with other non-RYR1
myopathies. This is a serious obstacle in the diagnostic process, where muscle biopsy is considered the “gold standard”.
Our cases illustrate the paradigm of some patients that in the
past would go years without a specific diagnosis, until they
developed histopathologic pathognomonic features. Genetic
testing has helped to solve many of these difficult diagnoses.
However, in the case of RYR1 genetic analysis, there are still
some difficulties to overcome specially when dealing with
sequence variants with unknown clinical significance. The
development of new approaches based on next generation
sequencing should increase the diagnostic yield in this field
and may bring some additional clues to the apparent genotype and/or phenotype discrepancies reported in some
patients.
We suggest that the predominance of type 1 fibers and
involvement of axial muscles may be an important element
to consider the RYR1 gene as candidate.
No funding was provided to conduct this study.
References
1. Quane KA, Healy JM, Keating KE, et al. Mutations in the ryanodine
receptor gene in central core disease and malignant hyperthermia.
Nat Genet. 1993;5:51-55.
2. Jungbluth H, Sewry CA, Muntoni F. Core myopathies. Semin Pediatr
Neurol. 2011;18:239-249.
3. Klein A, Suzanne L, Munteanu L, et al. Clinical and genetic findings in
a large cohort of patients with ryanodine receptor 1 gene-associated
myopathies. Human Mutation;00:1-8.
4. Amburgey K, Bailey A, Hwang JH, et al. Genotype-phenotype correlations in recessive RYR1-related myopathies. Orphanet J Rare Dis.
2013;8:117.
5. Dlamini N, Voermans NC, Lillis S, et al. Mutations in RYR1 are a
common cause of exertional myalgia and rhabdomyolysis. Neuromuscular Disorders. 2013;23:540-548.
6. Løseth S, Voermans NC, Torbergsen T, et al. A novel late-onset
axial myopathy associated with mutations in the skeletal muscle ryanodine receptor (RYR1) gene. J Neurol. 2013;260:
1504-1510.
7. Sewry CA, Muller C, Davis M, et al. The spectrum of pathology in
central core disease. Neuromuscul Disord. 2002;12:930-938.
8. Zhou H, Jungbluth H, Sewry CA, et al. Molecular mechanisms and
phenotypic variation in RYR1-related congenital myopathies. Brain.
2007;130:2024-2036.
9. Gambelli S, Malandrini A, Berti G, et al. Inheritance of a novel RYR1
mutation in a family with myotonic dystrophy type 1. Clin Genet.
2007;71:93-94.
10. Sato I, Wu S, Ibarra CA, et al. Congenital neuromuscular disease with
uniform type 1 fiber and RYR1 mutation. Neurology. 2008;70:
114-122.
11. Jungbluth H, Zhou H, Sewry CA, et al. Centronuclear myopathy
due to a de novo dominant mutation in the skeletal muscle
ryanodine receptor (RYR1) gene. Neuromuscul Disord. 2007;17:
338-345.
12. Wilmshurst JM, Lillis S, Zhou H, et al. RYR1 mutations are a common
cause of congenital myopathies with central nuclei. Ann Neurol.
2010;68:717-726. http://dx.doi.org/10.1002/ana.22119.
13. Kossugue PM, Paim JF, Navarro MM, et al. Central core disease due
to recessive mutations in RYR1 gene: is it more common than
described? Muscle Nerve. 2007;35:670-674.
14. Zhou H, Brockington M, Jungbluth H, et al. Epigenetic allele
silencing unveils recessive RYR1 mutations in core myopathies. Am J
Hum Genet. 2006;79:859-868.